The present invention relates to methods of monitoring, via polymerase chain reaction, the clinical progression of human immunodeficiency virus infection and its response to antiretroviral therapy. According to the invention, polymerase chain reaction assays may be used to predict immunological decline and to identify, at an early stage, patients whose infection has become resistant to a particular antiretroviral drug regimen.
Human immunodeficiency virus (HIV) isolated from patients treated with zidovudine (AZT) may demonstrate markedly reduced in vitro susceptibility to AZT (Larder et al., 1989, Science 243:1731-1734; Rooke et al., 1989, AIDS 3:411-415; Land et al., 1990, J. Infect. Dis. 161:326-329; Boucher et al., 1990, Lancet 336:585-590; Japour et al., 1991, Proc. Natl. Acad. Sci. 88:3092-96; Tudor-Williams et al., 1992, Lancet 339:15-19). This reduced susceptibility has been related to the duration of therapy with AZT and the severity of HIV disease at the time AZT therapy is begun (Richman et al., 1990, AIDS 3:743-756). Nucleotide sequence analysis of AZT-resistant HIV strains has revealed a number of mutations in the reverse transcriptase (RT) gene associated with decreased AZT susceptibility (Larder et al., 1989, Science 246:1155-1158; Larder et al., 1991, AIDS 5:4137-144; Kellam et al., 1992, Proc. Natl. Acad. Sci. USA 89:1934-1938; St. Clair et al., 1991, Science 253:1557-1559; Richman et al., 1991, J. Infect. Dis. 164:1075-1081). Molecular cloning experiments have confirmed that these mutations in the RT gene confer AZT resistance (Larder et al., 1989, Science 246:1155-1158; Larder et al., 1991, AIDS 5:137-144; Kellam et al., 1992, Proc. Natl. Acad. Sci. USA 89:1934-1938; St. Clair et al., 1991, Science 253:1557-1559). Of these mutations the one at codon 215 resulting in a single amino acid substitution (Thrxe2x86x92Tyr or Phe) has been shown to be the most common mutation and to have the greatest impact on in vitro susceptibility to AZT (Larder et al., 1991, AIDS 5:137-144; Richman et al., 1991, J. Infect. Dis. 164:1075-1081; Boucher et al., 1992, J. Infect. Dis. 165:105-110).
Several studies have addressed the relationship between in vitro AZT resistance, mutations in the RT gene and clinical disease. Richman and coworkers studied 32 patients with different stages of HIV disease and demonstrated that the development of in vitro AZT resistance was related to the duration of therapy with AZT and to the severity of disease at the time AZT was begun (Richman et al., 1990, AIDS 3:743-746). Boucher and coworkers studied HIV P24-antigenemic patients treated with AZT for 2 years. They observed that at 6 months, seven patients with a mutation at codon 215 had a weak, non-statistically significant trend toward lower CD4 counts compared to nine patients who were wild type at codon 215 (Boucher et al., 1990, Lancet 336:585-590). After 2 years nearly all patients had the mutation. Tudor-Williams and coworkers studied HIV isolates from 19 symptomatic children treated with AZT for 9-39 months and showed that in vitro AZT resistance was associated with poor clinical outcome (Tudor-Williams et al., 1992, Lancet 339:15-19).
Kahn and colleagues (Kahn et al., 1992, N. Engl. J. Med. 327:581-587) reported that patients infected with HIV who have relatively advanced disease and at least 16 weeks of previous zidovudine therapy may have clinical benefit if switched to didanosine monotherapy instead of remaining on zidovudine. The reason patients benefit from switching from zidovudine to didanosine is not fully understood, but one possibility is that dididanosine is suppressing zidovudine-resistant HIV.
Increasing evidence exists of a correlation between zidovudine-resistant HIV and disease progression in patients treated with zidovudine monotherapy (Tudor-Williams et al., 1992, Lancet 339:15-19; St. Clair et al., 1993, AIDS 6:891-897; Kozal et al., 1993, J. Infect. Dis. 167:526-532; Montaner et al., 1993, AIDS 7:189-196). It has been shown that HIV can also develop resistance to didanosine (ddI)(St. Clair et al., 1991, Science 253:1557-1559; Reichman et al., 1993, Antiviral Res. 20:267-277; Japour et al., 1991, Proc. Natl. Acad. Sci. USA 88:3092-3096). As with the resistance of HIV to zidovudine, the decrease in susceptibility of HIV to didanosine has been shown to be caused by specific mutations in the HIV reverse-transcriptase gene. St. Clair and colleagues (St. Clair et al., 1991, Science 253:1557-1559) identified the first mutation in the reverse-transcriptase gene to confer resistance to didanosine, a mutation at codon 74 that results in an amino acid change from leucine to valine. The codon 74 mutation in an HIV construct can induce an eightfold decrease in susceptibility to didanosine.
Although other mutations have been reported to confer didanosine resistance (Gu et al., 1992, J. Virol. 66:28-35), most data to date suggest the codon 74 mutation is the primary mutation responsible for didanosine resistance in patients receiving didanosine monotherapy.
The present invention relates to methods of monitoring, via polymerase chain reaction (PCR), the clinical progression of human immunodeficiency virus (HIV) infection and its response to antiviral therapy. It is based, in part, on the discovery that plasma HIV RNA copy number, as measured using PCR, may be used as a sensitive marker of the circulating HIV viral load to assess the therapeutic effect of antiretroviral compounds. In working examples described herein, an increase in plasma HIV RNA copy number was found to correlate with disease progression, and successful antiretroviral therapy was found to correlate with a decline in plasma HIV RNA copy number.
The invention is also based, in part, on the discovery that genetic changes in HIV which confer resistance to antiretroviral therapy may be rapidly determined directly from patient peripheral blood mononuclear cells (PBMC) and/or plasma HIV RNA using a xe2x80x9cnestedxe2x80x9d PCR procedure. In working examples disclosed herein, a mutation at codon 215 of HIV reverse transcriptase (RT) was found to occur in AZT-treated patients which correlated with refractoriness to AZT treatment. The mutation was found in plasma HIV RNA one to eight months before it was detectable in PBMC. The development of the codon 215 mutation in HIV RT was found to be a harbinger of immunological decline, which occurred between six and twelve months after the mutation was detectable in plasma HIV RNA.
In particular embodiments of the invention, PCR assay may be used to monitor the clinical progression of HIV infection in patients receiving antiretroviral therapy. An increase in plasma HIV copy number detected by such an assay would correlate with refractoriness to treatment. If a patient being treated with an antiretroviral therapeutic agent exhibits an increase in plasma HIV RNA copy number, a physician should consider altering the patients treatment regimen. It should be noted that the present invention offers the advantage of being more sensitive in measuring HIV virus than standard methods which measure plasma p24 antigen or infectious virus detectable by culture techniques.
In further embodiments of the invention, PCR assay may be used to detect mutations at codon 215 of HIV RT which correlate with resistance to antiretroviral therapy and which precede immunologic decline by 6-12 months. Once mutation at codon 215 has been detected in a patient undergoing antiretroviral therapy, an alteration in the therapeutic regimen must be considered. The speed at which a modified regimen should be instituted may depend on whether the mutation is present in plasma HIV RNA or PBMC. If the mutation is present in PBMC, a rapid alteration in therapy may be warranted.
In further embodiments of the invention, PCR assay may be used to detect and monitor the absolute concentrations and relative proportions of virus which mutations at codon 74 of HIV RT, a mutation which correlates with resistance to therapy with didanosine (ddI). When mutation at codon 74 has been detected in patient undergoing monotherapy with ddI, an alteration in the therapeutic regimen must be considered. Such alteration may include combination therapy, e.g. ddI and AZT, or combination therapy with another antiviral agent.
In patients suffering from HIV infection, opportunistic infections arising as a result of a compromised immune system can be rapidly fatal. It is therefore extremely important to strive to avoid deterioration of the immune system in these patients. Because the present invention enables the early prediction of immunological decline, it allows alteration of a patient""s therapeutic regimen so as to avoid opportunistic infections, and therefore may be used to promote survival and improve the quality of life of HIV-infected patients.